The present invention relates to assays and analyses of genes or DNAs relating to gene diagnoses, gene therapies, and production of various substances by genes, or the like. In particular, the present invention relates to improvement of pyrosequencing and systems thereof, namely DNA base sequencing methods or sequence monitoring methods and DNA base sequencing systems or sequence monitoring systems.
The progress of the human genome-mapping project is prompting a trend towards conducting diagnoses of diseases and producing useful novel substances at the gene level by reading DNA sequences. However, a big problem remains to be solved: how to quickly analyze a large amount of DNA samples. Although fundamental DNA base sequence data have been almost completely revealed by the genome mapping, it is necessary to compare sequences of various samples with those of standard samples previously sequenced, in order to study sequences of these DNAs and gene functions. Therefore, a large number of various samples have to be quickly sequenced to find differences between the sequences and examine their correlations with biological functions, although the length of the DNAs to be sequenced at a time may be small.
Conventionally, DNAs are sequenced by a DNA base sequencing method using gel electrophoresis, and a DNA sequencer is commercially available and widely used as an apparatus. Recently, a speedy sequencing method is drawing attention, in which a sample DNA is hybridized with a DNA probe using a DNA chip, in which many kinds of DNA probes are immobilized on a solid cell to make a probe array.
On the other hand, a sequencing method different from the above-mentioned methods, called pyrosequencing, has been proposed. In the pyrosequencing method, DNA complementary strand synthesis is monitored to determine sequences, namely, pyrophosphate released as a reaction product upon synthesizing a complementary DNA strand is converted into ATP, which reacts with luciferin using luciferase to generate luminescence. Since pyrosequencing is inexpensive and can be used for sequencing a large number of samples simultaneously, it is a promising method as a high throughput monitor for DNA.
A reported pyrosequencing is briefly explained as follows. The apparatus used is a so-called luminescence photometer. Reagents, including DNA samples; primers to determine the starting point of complementary strand synthesis; DNA synthesizing enzymes; an enzyme apyrase to decompose dNTP (deoxynucleotide triphosphates) which has been added as a substrate and remained unreacted; sulfurylase to convert pyrophosphate into ATP; luciferin; and luciferase involved in the reaction of luciferin with ATP, are placed in a titer plate. At this moment, no complementary strand synthesis occurs because dideoxynucleotides (ddNTPs), a substrate for the reaction, is not present. Four kinds of ddNTPs (i.e., dATP, dCTP, dTTP and dGTP) are added in a designated order by an ink jet system. If dCTP is the designated base to be synthesized, no reaction occurs when dATP, dTTP or dGTP is added. Reaction occurs only when dCTP is added, then the complementary strand is extended by one base length, and pyrophosphate (PPi) is released. This pyrophosphate is converted into ATP by ATP sulfurylase and the ATP reacts with luciferin in the presence of luciferase to emit chemiluminescence. This chemiluminescence is detected using a secondary photon multiplier tube or the like. Remaining dCTP or unreacted dNTP is decomposed by apyrase which converts it into a form which has no effect on the subsequent repetitive dNTP injection and the reaction which follows. The four kinds of dNTP are added repeatedly in a designated order and the base sequence is determined one by one according to the presence or absence of chemiluminescence emitted each time. This series of reactions are shown in FIG. 3 (see Ronaghi, M. et al., Science 281, 363-365 (1998)).
One problem in the current pyrosequencing method, in which ink jet nozzles are used for dNTP injection, is that it requires a considerably large space for the apparatus including a control part for the ink jet nozzles or the like. Another problem is that the target DNA to be provided as a sample in the reaction vessel has to be a single strand, which requires extra labor for the sample preparation. Further, sequencing is not possible in the presence of DNAs, which undergo complementary strand synthesis, other than the target DNA. The reported possible length of DNA to be sequenced ranges between 20 bases and 30 bases. This is because the sequencing is involved in a step reaction, in which the efficiency of the reaction is largely affected by the possible length of the base to be sequenced.
Examples of possible systems in which pyrosequencing is used include a palm-sized DNA sequencer, a DNA sequencer for large scale analyses for gene diagnoses or comparative analyses, and a DNA mutation analysis system. However, for practical use of these systems, several technical problems remain to be solved: (1) how to implement a simple and inexpensive compact apparatus, (2) how to minimize the time required for sample preparation, and how to analyze various samples simultaneously in a simple way, and (3) how to uniformly carry out reactions to increase the reaction efficiency. Accordingly, a compact, inexpensive apparatus and a system which effects simpler sample preparation and sequencing of longer base sequences, if necessary, are needed.
Objects of the present invention are to provide a compact, simple and convenient DNA base sequencing system or a sequence monitoring apparatus, and to provide a DNA base sequencing method or a sequence monitoring method in which sample preparation can be carried out in a simple and easy manner.